Prenylfavanone compounds and uses thereof

ABSTRACT

The present invention relates to new prenylflavanone compounds and a pharmaceutical composition comprising at least one of the compounds.

BACKGROUND OF THE INVENTION

The preset invention relates to new prenylflavanone compounds and usesthereof.

Propolis is a resinous mixture that honeybees collect from tree buds,fruits, sap flows, or other botanical sources. It has been reported topossess various active components and exhibit a broad spectrum ofbiological activities, including antitumor⁽³⁾, antioxidant⁽⁴⁾,antibacterial⁽⁵⁾, antiviral⁽⁶⁾, antifungal⁽⁷⁾, and anti-inflammatoryactivities⁽⁸⁾. Propolis from different areas may contain differentactive components.

We previously reported eight prenylflavanones isolated from Taiwanesepropolis which are represented by Formulae 1 to 8 as below,respectively.

These propolins have been reported to exhibit a broad spectrum ofbiological activities, including anticancer⁽¹⁰⁻¹⁵⁾, antioxidant⁽¹⁰⁻¹⁶⁾and antimicrobial activities⁽¹⁶⁾. In addition, recent studiesdemonstrated that Okinawan propolis contained active components similarto that of Taiwanese propolis⁽¹⁷⁻¹⁸⁾.

Histone deacetylase (HDAC) is an enzyme that catalyses deacetylation ofthe ε-amino group of lysine amino acid residues in the N-terminal tailsof histones. Some HDAC inhibitors have been found and demonstrated tohave therapeutic efficacy on patients suffered from different types ofcancers in preclinical and clinical stages⁽¹⁹⁻²¹⁾. According to thecurrent model for the anticancer mechanism of HDAC inhibitors, theinhibitors induce hyperacetylation of core histones and thus triggerchromatin remodeling and wake up silent genes such as tumor suppressorgenes which result in inhibition of tumor cells growth⁽²⁶⁻²⁷⁾.

There is still a need to find new components from the multi-functionalpropolis and evaluate their useful physiological activities.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a new prenylflavanonecompound which is represented by a formula selected from the groupconsisting of:

In another aspect, the present invention provides a pharmaceuticalcomposition comprising at least one of the prenylflavanone compounds asdescribe above.

In yet another aspect, the present invention to provide a method fortreating a disease or condition or providing a desired effect in asubject in need thereof comprising administrating to the subject atleast one of the compounds as described above or the above-mentionedpharmaceutical composition. Particular embodiments are described below.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the preferred embodiments shown.

In the drawings:

FIG. 1 represents the key HMBC correlations between H and C in PropolinI.

FIG. 2 represents the key HMBC correlations between H and C in PropolinJ.

FIG. 3 shows the peaks of the reversed-phase preparative HPLC in Example2.

FIG. 4(A) shows the staining results of MCF-7 cells treated withdifferent propolins at various concentrations for 24 hours, wherein (a)refers to the control treatment; (b), (c) and (d) refer to the treatmentby Propolin F at a concentration of 5, 10 and 15 μg/mL, respectively;(e), (f) and (g) refers to the treatment by Propolin I at aconcentration of 2.5, 5.0 and 7.5 μg/mL, respectively; and (h), (i) and(j) refer to the treatment by Propolin J at a concentration of 5, 10 and15 μg/mL.

FIG. 4(B) shows the staining results of MDA-MB-231 cells treated withdifferent propolins at various concentrations for 24 hours, wherein (a)refers to the control treatment; (b), (c) and (d) refer to the treatmentby Propolin F at a concentration of 5, 10 and 15 μg/mL, respectively;(e), (f) and (g) refers to the treatment by Propolin I at aconcentration of 2.5, 5.0 and 7.5 μg/mL, respectively; and (h), (i) and(j) refer to the treatment by Propolin J at a concentration of 5, 10 and15 μg/mL, respectively.

FIG. 4(C) shows the staining results of MCF-7 cells treated withdifferent propolins at various concentrations for 72 hours, wherein (a)refers to the control treatment; (b), (c) and (d) refer to the treatmentby Propolin F at a concentration of 5, 10 and 15 μg/mL, respectively;(e), (f) and (g) refer to the treatment by Propolin I at a concentrationof 2.5, 5.0 and 7.5 μg/mL, respectively; and (h), (i) and (j) refer tothe treatment by Propolin J at a concentration of 5, 10 and 15 μg/mL,respectively.

FIG. 4(D) shows the staining results of MDA-MB-231 cells treated withdifferent propolins at various concentrations for 72 hours, wherein (a)refers to the control treatment; (b) and (c) refer to the treatment byPropolin F at a concentration of 10 and 15 μg/mL, respectively; and (d)and (e) refer to the treatment by Propolin I at a concentration of 5.0and 7.5 μg/mL, respectively; and (f) and (g) refer to the treatment byPropolin J at a concentration of 10 and 15 μg/mL, respectively.

FIG. 4(E) shows the numbers of MCF-7 cells treated with differentpropolins at various concentrations for 72 hours.

FIG. 5(A) shows the result of the flow cytometric assay for the MCF-7cells treated with different propolins at various concentrations for 72hours, wherein (a) refers to the control treatment; (b), (c) and (d)refer to the treatment by Propolin F at a concentration of 5, 10 and 15μg/mL, respectively; (e), (f) and (g) refers to the treatment byPropolin I at a concentration of 2.5, 5.0 and 7.5 μg/mL, respectively;and (h), (i) and (j) refer to the treatment by Propolin J at aconcentration of 5, 10 and 15 μg/mL.

FIG. 5(B) shows the result of the flow cytometric assay for theMDA-MB-231 cells treated with different propolins at variousconcentrations for 24 hours, wherein (a) refers to the controltreatment; (b), (c) and (d) refer to the treatment by Propolin F at aconcentration of 5, 10 and 15 μg/mL, respectively; (e), (f) and (g)refers to the treatment by Propolin I at a concentration of 2.5, 5.0 and7.5 μg/mL, respectively; and (h), (i) and (j) refer to the treatment byPropolin J at a concentration of 5, 10 and 15 μg/mL.

FIG. 5(C) shows the result of the flow cytometric assay for theMDA-MB-231 cells treated with different propolins at variousconcentrations for 72 hours, wherein (a) refers to the controltreatment; (b) and (c) refer to the treatment by Propolin F at aconcentration of 10 and 15 μg/mL, respectively; and (d) and (e) refer tothe treatment by Propolin I at a concentration of 5.0 and 7.5 μg/mL,respectively; and (f) and (g) refer to the treatment by Propolin J at aconcentration of 10 and 15 μg/mL, respectively.

FIG. 6(A) shows the results of the immunocytochemistry study in Example7.

FIG. 6(B) shows the results of the western blotting assay in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as is commonly understood by one of skill in theart to which this invention belongs.

In one aspect, the present invention provides a prenylflavanone compoundisolated from Taiwanese propolis.

One embodiment of the prenylflavanone compound is5,7,3′,5′-tetrahydroxy-6-farnesylflavanone (named “Propolin I”), whichis represented by the

The compound of Formula I has the molecular formula of C₃₀H₃₆O₆, and amolecular weight of 492.25 Da.

Another embodiment of the prenylflavanone compound is5,7,4′-trihydroxy-6-geranylflavanone (named “Propolin J”), which isrepresented by the following formula:

The compound of Formula II has the formula of C₂₅H₂₈O₅ and a molecularweight of 408.19 Da.

The above-mentioned prenylflavanone compounds of the invention haveinhibitory effects on HDAC enzymatic activity and growth of cancercells, preferably that of breast cancer cells.

Accordingly, in another aspect, the present invention provides apharmaceutical composition as a HDAC inhibitor comprising at least oneof the compounds of the invention and a pharmaceutical acceptableexcipient or carrier.

In yet another aspect, the present invention provides a pharmaceuticalcomposition for inhibiting cancer cell growth comprising at least one ofthe compounds of the invention and a pharmaceutically acceptableexcipient or carrier.

The present invention also provides a pharmaceutical composition fortreating a disease in association with histone deacetylation comprisingat least one of the compounds of the invention and a pharmaceuticallyacceptable excipient or carrier.

In addition, many of known HDAC inhibitors have been demonstrated tohave neuroprotective effects^((30,31)), implying that a HDAC inhibitoris useful in the treatment of neurodegenerative diseases. Examples ofthe neurodegenerative diseases include, but are not limited to, multiplesclerosis (MS)⁽³²⁾, Huntington's disease (HD)^((33,39,40)), spinalmuscular atrophy (SMA)^((34,35,36)), spinal and bullar muscular atrophy(SBMA)⁽³⁷⁾, and amyotrophic lateral sclerosis (ALS)⁽³⁸⁾.

Accordingly, the present invention thus relates to a pharmaceuticalcomposition having neuroprotective effects comprising at least one ofthe compounds of the invention and a pharmaceutically acceptableexcipient or carrier.

The present invention also relates to a pharmaceutical composition fortreating a neurodegenerative disease comprising at least one of thecompounds of the invention and a pharmaceutically acceptable excipientor carrier. Preferably, the neurodegenerative disease is multiplesclerosis (MS), Huntington's disease (HD), spinal muscular atrophy(SMA), spinal and bullar muscular atrophy (SBMA), or amyotrophic lateralsclerosis (ALS).

In making the composition of the invention, the compound of theinvention as the active ingredient is usually diluted with an excipientor enclosed within a carrier which can be in the form of a capsule,sachet, paper or other container. The excipient employed is typically anexcipient suitable for administration to human subjects or othermammals. Some examples of suitable excipients include, but are notlimited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches,gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,water, syrup, and methyl cellulose. In addition, the composition of theinvention can be in the form of tablets, pills, powders, suspensions,emulsions, solutions, syrups, aerosols (as a solid or in a liquidmedium), ointments, capsules, or sterile packaged powders.

The compositions can be administered to a subject including humans,monkeys, dogs, etc. by an oral route, for example, or they can beadministered by a parenteral route in the form of injectablepreparations. Typically the composition of the invention contains fromabout 125 to about 250 mg of the compound of the invention (Propolins Ior J, or both). The dose for adults is preferably between about 500 andabout 1000 mg per day, which can be administered in a single dose or inthe form of separated doses.

In another aspect, the present invention provides a method for treatinga disease or condition or providing a desired effect in a subject inneed thereof comprising administrating to the subject at least one ofthe compounds as described above or the above-mentioned pharmaceuticalcomposition.

As used herein, a subject in need of the treatment according to theinvention includes human and non-human mammals. Non-human mammalsinclude, but are not limited to, companion animals such as cats, dogsand the like and farm animals such as cattle, horses, sheep, goats,swine and the like.

In particular, the above-mentioned method of the invention is useful ininhibiting cancer cell growth in a subject. More particular, the methodof the invention is useful in treating growth of breast cancer cells ina subject.

In addition, the method of the invention is useful in treating a diseasein association with histone deacetylation. Moreover, the method of theinvention can provide neuroprotective effects in a subject.

Furthermore, the method of the invention is useful in treating aneurodegenerative disease in a subject. Specifically, theneurodegenerative disease is selected from the group consisting ofmultiple sclerosis (MS), Huntington's disease (HD), spinal muscularatrophy (SMA), spinal and bullar muscular atrophy (SBMA), andamyotrophic lateral sclerosis (ALS).

The present invention will now be described more specifically withreference to the following embodiments, which are provided for thepurpose of demonstration rather than limitation.

EXAMPLE 1 Primary Extraction

200 g of Taiwanese propolis of TW-I grade (collected from hives locatedin Tainan, Taiwan)⁽²⁹⁾ was homogenized by stirring at a low temperature.The homogenized sample was then washed 3 times with 1.0 L of deionizedwater and the residue was extracted 3 times with 95% ethanol. Thefiltered ethanol extract was evaporated to dryness under reducedpressure and a brown powder was obtained (135.6 g) which was stored at−20° C. until further purification.

EXAMPLE 2 Isolation and Purification of Compounds

The brown powder obtained from the ethanol extract was dissolved inmethanol and applied to a Sephadex LH-20 column (Amersham PharmaciaBiotech AB, Uppsala, Sweden) using 95% ethanol as the eluting solvent.All the elutes, including the fractions obtained by the follow-upchromatographies, were assayed for their effects on human breast cancerproliferation, and the active fractions were again separated bychromatographies on a Sephadex LH-20 column using 95% ethanol as theeluting agent. Next, the active fractions were subjected to silica gelcolumn chromatography (Kiesel gel 60, E. Merck, Darmstadt 1, Germany)using an n-hexane/EtOAc solvent system.

The purification of the most active fraction was performed byreversed-phase preparative HPLC (n-hexane/EtOAc, 30:70). Theexperimental conditions were as follows: the column was Luna Phenomenex(C 18, 250×4.6 mm); the solvent system was methanol/water (8:2); theflow rate was 1 mL/min; and the detection was conducted at UV 280 nm.Fractions at the retention time, 19.0 min (Propolin I) and 10.5 min(Propolin J), were collected whereby Propolin I (light yellow powder)and Propolin J (light yellow liquid) were isolated, respectively. FIG. 3shows the peaks of the reversed-phase preparative HPLC.

EXAMPLE 3 Identification of Propolin I and Propolin J

The structure of each of Propolins I and J was analyzed by using thefollowing instruments: Perkin Elmer 1760-X IR-FT spectrometer; Hitachi150-20 UV; Jasco J-710 spectropolarimeter; Finnigan MAT-95XL massspectrometer (EI); and Bruker AV-500 spectrometer using the solvent peak(MeOH-d3) as a reference standard.

Propolin I

The physicochemical data of Propolin I is shown as follows:

[┐]²⁰ _(D)+3.8 (c 0.26, CH₃OH); IR νmax (film) 3747, 3390, 2923, 1636cm⁻¹; UV (EtOH) λmax nm (log ε) 207.0 (1.65), 291 (0.73); CD (MeOH) 347nm (Δε+3.19), 294 nm (Δε−3.98); HREIMS m/z 492.2512 (calcd for C₃₀H₃₆O₆492.2512); ¹H and ¹³C NMR.

From the ¹H NMR spectrum, it was confirmed that there were four olefinicmethyl groups (δ_(H) 1.52, 1.56, 1.63, 1.74), eight methylene protons(δ_(H) 1.87, 1.95, 2.05), two benzylic methylene protons (δ_(H) 3.20)and two vinyl protons (δ_(H) 5.04, 5.18), which indicated the presenceof a farnesyl group. Besides, the ¹³C NMR spectrum showed that therewere four methyl groups (δ_(c), 16.1, 16.2, 17.8, 25.9), five methylenecarbons (δ_(c), 21.8, 27.3, 27.8, 40.8, 40.9), three tertiary olefiniccarbons (δ_(c) 124.1, 125.3, 125.6) and three quaternary olefiniccarbons (δ_(c) 132.0, 134.9, 135.8) which further supported the presenceof a farnesyl group. Moreover, the ABX system at δ_(H) 5.21 (1H, dd,J=3.0, 13.0 Hz), 2.65 (1H, dd, J=3.0, 17.0 Hz), and 3.02 (1H, dd,J=13.0, 17.0 Hz) displayed a characteristic pattern for a flavanoneskeleton at the H-2 and H-3 position, respectively.

In addition, the total ¹H and ¹³C NMR assignments and connectivitieswere determined based on the inference of the ¹H—¹H COSY, HSQC, and HMBCdata. The HMBC spectrum of Propolin I revealed that the methylene signalat δ_(H) 3.20 (H-1″) was correlated with C-5 (δ_(c) 162.5) and C-7(δ_(c) 165.9), respectively, which suggested that the farnesyl group wasattached to C-6 (FIG. 1). In addition, its CD spectra indicated that theconfiguration of C-2 was S-form.

Accordingly, Propolin I was identified as5,7,3′,5′-tetrahydroxy-6-farnesylflavanone, which is represented byFormula I as mentioned above. This compound was isolated for the firsttime and had not been disclosed in published literatures.

Propolin J

The physicochemical data of Propolin J is shown as follows:

[□]²⁰ _(D)+2.4 (c 0.41, CH₃OH); IR νmax (film) 3747, 3393, 2925, 2361,1637 cm⁻¹; UV (EtOH) λmax nm (log ε) 209.0 (1.82), 293 (0.92); CD (MeOH)328.9 nm (Δε +2.37), 289 nm (Δε−3.98); HREIMS m/z 408.1944 (calcd forC₂₅H₂₈O₅ 408.1937); ¹H and ¹³C NMR.

From the ¹H NMR spectrum, it was confirmed that there were threeolefinic methyl groups (δ_(H) 1.55, 1.61, 1.74), four methylene protons(δ_(H) 1.93, 2.04), two benzylic methylene protons (δ_(H) 3.18) and twovinyl protons (δ_(H) 5.05, 5.18), which indicated the presence of ageranyl group. Besides, the ¹³C NMR spectrum shown that there were threemethyl groups (δ_(c) 16.2, 17.7, 25.9), three methylene carbons (δ_(c)21.8, 27.7, 40.9), two tertiary olefinic carbons (δ_(c) 123.9, 125.5),and two quaternary olefinic carbons (δ_(c) 132.0, 135.3), which furthersupported the presence of a geranyl group. Moreover, the ABX system atδ_(H) 5.29 (1H, dd, J=2.7, 13.0 Hz), 2.67 (1H, dd, J=2.7, 17.0 Hz), and3.08 (1H, dd, J=13.0, 17.0 Hz) displayed a characteristic pattern for aflavanone skeleton at H-2 and H-3 positions, respectively.

In addition, the total ¹H and ¹³C NMR assignments and connectivitieswere determined based on the inference of the ¹H—¹H COSY, HSQC, and HMBCdata. The HMBC spectrum of Propolin J revealed that the methylene signalat δ_(H) 3.18 (H-1″) correlated with C-5 (δ_(c) 162.5) and C-7 (δ_(c)166.0), which suggested that the geranyl group was attached to C-6 (FIG.2). In addition, its CD spectra indicated that the configuration of C-2was S-form.

Accordingly, Propolin J was identified as5,7,4′-trihydroxy-6-geranylflavanone, which is represented by Formula IIas mentioned above. This compound was isolated for the first time andhad not been disclosed in published literatures.

Table 1 lists the physicochemical data of Propolins I and J.

TABLE 1 Propolin I Propolin J position δ_(C), multi. δ_(H) (J in Hz)δ_(C), multi. δ_(H) (J in Hz) 2 80.5, CH 5.21, dd (3.0, 13.0) 80.4, CH5.29, dd (2.7, 13.0) 3 44.3, CH₂ 2.65, dd (3.0, 17.0) 44.2, CH₂ 2.67, dd3.02, dd (13, 17.0) (2.7, 17.0) 3.08, dd (13, 17.0) 4 197.8, qC 197.9,qC 5 162.5, qC 162.5, qC 6 109.7, qC 109.7, qC 7 165.9, qC 166.0, qC 895.4, CH 5.92, s 95.4, CH 5.93, s 9 162.4, qC 162.4, qC 10  103.2, qC103.2, qC  1′ 131.9, qC 131.2, qC  2′ 114.7^(a), CH 6.90, s 129.0, 7.30,d (8.6) CH  3′ 146.5^(b), qC 116.3, 6.80, d (8.6) CH  4′ 116.2, CH 6.77,s 159.0, qC  5′ 146.8^(b), qC 116.3, 6.80, d (8.6) CH  6′ 119.2^(a), CH6.77, s 129.0, 7.30, d (8.6) CH  1″ 21.8, CH₂ 3.20, d (7.2) 21.8, CH₂3.18, d (7.1)  2″ 124.1, CH 5.18, t (5.1) 123.9, 5.18, t (7.1) CH  3″134.9, qC 135.3, qC  4″ 16.1, CH₃ 1.74, s 16.2, CH₃ 1.74, s  5″ 40.8,CH₂ 1.95, m 40.9, CH₂ 1.93, m  6″ 27.3, CH₂ 2.05, dd (6.9, 14.0) 27.7,CH₂ 2.04, dd (6.9, 14.9)  7″ 125.3, CH 5.04, m 125.5, 5.05, t (7.2) CH 8″ 135.8, qC 132.0, qC  9″ 16.2, CH₃ 1.52, s 25.9, CH₃ 1.61, s 10″40.9, CH₂ 1.87, m 17.7, CH₃ 1.55, s 11″ 27.8, CH₂ 1.95, m 12″ 125.6, CH5.04, m 13″ 132.0, qC 14″ 25.9, CH₃ 1.63, s 15″ 17.8, CH₃ 1.56, s^(a,b)Data interchangeable.

EXAMPLE 4 Cell Culture and Cytotoxicity Assay

Human breast cancer MCF-7 and MDA-MB-231 cells were purchased from theFood Industry Research and Development Institute (Hsinchu, Taiwan). Thecells were cultured in Dulbecco's modified Eagle's medium (Gibco)containing 10% fetal bovine serum (FBS), a 1% dilution ofpenicillin-streptomycin, and 2 mM glutamine. Cells were maintained at37° C. in a humidified atmosphere of 95% air and 5% CO₂. However,MDA-MB-231 cells were cultured in L-15 medium (Gibco) containing 10%fetal bovine serum (FBS), a 1% dilution of penicillin-streptomycin, and2 mM glutamine. Cells were maintained at 37° C. in a humidifiedatmosphere of 100% air and 0% CO₂.

The propolins F, I, and J were dissolved in dimethyl sulfoxide (DMSO)and prepared at a fixed concentration of 10 mg/mL. The cells (1.5×10⁶per dish) were cultured in a 100-mm dish and incubated for 14 h prior totreatment with DMSO or with different concentrations of propolins (2.5,5.0, 10, and 20.0 μg/mL) for 48 h. The propolins were small compoundswith their MWs ranging from 408-492 Da. These values were converted totheir respective molarities.

The cells were counted and their viability was determined by trypan blueexclusion assay. The activity was shown as IC₅₀ value (the concentrationrequired to induce 50% cytotoxicity), and the average value was obtainedfrom triplicate data points.

Table 2 shows the IC₅₀ (μM) values of Propolins I and J of the inventionand that of Propolin F as a control obtained in the cytotoxic assay.

TABLE 2 Compounds IC₅₀ values^(a) Propolin F 47.2 Propolin I 10.2Propolin J 36.8 ^(a)IC50 (μM) values were from one representativeexperiment of three independent experiments.

Accordingly, the compounds of the invention (Propolins I and J) havebeen found to have cytotoxic activity against human breast cancer cells.

EXAMPLE 5 Inhibitory Effect of Propolins I and J on Growth of CancerCells

Cell Staining

Two types of breast cancer cell lines, MCF-7 (ER receptor positive) andMDA-MB-231 cells (ER receptor negative), were cultured in the conditionsas described in Example 5. The cells were treated with differentpropolins at various concentrations for 24 or 72 hours. The treatedcells were stained with trypan blue and evaluated for viability.

FIG. 4(A) shows the staining results of MCF-7 cells treated with thepropolins at various concentrations for 24 hours. FIG. 4(B) shows thestaining results of MDA-MB-231 cells treated with the propolins atvarious concentrations for 24 hours. FIG. 4(C) shows the stainingresults of MCF-7 cells treated with the propolins at variousconcentrations for 72 hours. FIG. 4(D) shows the staining results ofMDA-MB-231 cells treated with the propolins at various concentrationsfor 72 hours. FIG. 4(E) shows the cell numbers of MCF-7 cells treatedwith the propolins at various concentrations for 72 hours.

Flow Cytometric Analysis

Human breast cancer MCF-7 and MDA-MB-231 cells (1.5×10⁶) in a 100-mmdish were treated with various concentrations of propolins F, I, and Jfor 24 or 72 h. Cells were trysinized and collected with ice cold PBS.The cells were resuspended in 200 μL PBS and fixed by adding 800 μL oficed 100% ethanol then incubated overnight at −20° C. The cell pelletswere collected by centrifugation, resuspended in 1 mL of hypotonicbuffer (0.5% Triton X-100 in PBS and 1 μg/mL RNase A), and incubated at37° C. for 30 min. Then, 1 mL of PI solution (50 μg/mL) was added, andthe mixture was allowed to stand at 4° C. for 30 min. Cellular DNAcontent was then analysed by FACScan cytometry (Becton Dickinson).

FIG. 5(A) shows the result of MCF-7 cells treated with the propolins atvarious concentrations for 72 hours. FIG. 5(B) shows the result ofMDA-MB-231 cells treated with the propolins at various concentrationsfor 24 hours. FIG. 5(C) shows the result of MDA-MB-231 cells treatedwith the propolins at various concentrations for 24 hours.

As shown in FIGS. 4 and 5, the compounds of the invention weredemonstrated having inhibitory effect on growth of the cancer cells.

Western Blotting Assay

Human breast cancer MCF-7 cells (1.5×10⁶) on 100-mm dishes were treatedwith propolins F, I, and J at various concentrations for 24 h. Aftertreatment, cells were collected and resuspended in 100 μl lysis buffer.Equal amounts of proteins (30 μg), were mixed with 2×sample buffer andresolved by 12.5% SDS-PAGE for β-actin, Bid, p21, Ac-histone 3, CTPS,and gelsolin detection. Proteins were electrotransferred to an immobilonmembrane (PVDF; Millipore Corp.), and equivalent protein loading wasverified by staining the membrane with reversible dye amido black (SigmaChemical Co.). This was followed by overnight blocking with a solutioncomposed of 20 mM Tris-HCl (pH 7.4), 125 mM NaCl, 0.2% Tween 20, and 3%BSA. Specific antibodies used were anti-human Bid (1:500 of rabbitpolyclonal; Cell Signaling Technology, Inc.), anti-Ac-histone 3 (1:1000of rabbit polyclonal; Cell Signaling Technology, Inc.), anti-p21 (1:1000of mouse monoclonal; BD Pharmingen Technology, Inc.), anti-CTPS (1:1000of mouse monoclonal; ABNOVA TAIWAN Corporation), gelsolin (1: 1000 ofmouse monoclonal; Sigma Chemical Co.), and anti-β-actin (1:5000 of mousemonoclonal; Cell Signaling Technology, Inc.). These proteins weredetected by chemiluminescence (ECL, Amersham).

EXAMPLE 6 HDAC Enzymatic Activity Assay

Immunocytochemistry Study

MCF-7 cells were cultured on six-well of culture slides and treated withpropolins F, I, and J for 6 h. SAHA known as a HDAC inhibitor was usedas a positive control. Slides were fixed for 30 minute at roomtemperature in a solution of 80% methanol and then washed 3 times inPBS. Cells were permeabilized for 30 minutes at room temperature with0.3% Triton X-100, and then blocked in 10% fetal bovine serum (FBS) inPBS for 1 hour at room temperature before incubating overnight at 4° C.with antiacetylated histone H3 (Cell Signaling) diluted 1:500. Afterwashing 3 times in PBS, the slides were stained with antirabbitIgG-conjugated secondary antibody for 1.0 hours then washed 3 times inPBS, and mounted with mounting medium (Sigma). In a parallel controlexperiment, it was observed that omission of either primary antibodyeliminated staining. Slides were analyzed by means under a fluorescencemicroscope with an Olympus PM30 camera (Melville, N.Y.). FIG. 6(A) showsthe results of the immunocytochemistry study.

Western Blotting Assay

The western blotting assay was carried out for Ac-histone 3, p21,gelsolin, CTPS and β-actin according to the method as described inExample 6. FIG. 6(B) shows the result of the western blotting assay.

As shown in FIGS. 6(A) and (B), the compounds of the invention wereproved having inhibitory effects on HDAC enzymatic activity.

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1. A compound which is represented by a formula selected from the groupconsisting of:


2. A pharmaceutical composition comprising at least one of the compoundsaccording to claim 1 and a pharmaceutical acceptable excipient orcarrier.
 3. The pharmaceutical composition of claim 2, which is usefulas a histone deacetylase inhibitor.
 4. The pharmaceutical composition ofclaim 2, which is useful in inhibiting cancer cell growth.
 5. Thepharmaceutical composition of claim 2, which is effective in inhibitinggrowth of breast cancer cells.
 6. The pharmaceutical composition ofclaim 2, which is useful in treating a disease in association withhistone deacetylation.
 7. The pharmaceutical composition of claim 2,which can provide neuroprotective effects.
 8. The pharmaceuticalcomposition of claim 2, which is useful in treating a neurodegenerativedisease.
 9. The pharmaceutical composition of claim 8, wherein theneurodegenerative disease is selected from the group consisting ofmultiple sclerosis (MS), Huntington's disease (HD), spinal muscularatrophy (SMA), spinal and bullar muscular atrophy (SBMA), andamyotrophic lateral sclerosis (ALS).
 10. A method for inhibiting cancercell growth in a subject in need thereof, comprising administeringadministrating to the subject a compound according to claim 1 or apharmaceutical composition according to claim
 2. 11. A method forinhibiting growth of breast cancer cells in a subject in need thereof,comprising administering administrating to the subject a compoundaccording to claim 1 or a pharmaceutical composition according to claim2.
 12. A method for treating a disease in association with histonedeacetylation in a subject in need thereof, comprising administeringadministrating to the subject a compound according to claim 1 or apharmaceutical composition according to claim
 2. 13. A method forproviding neuroprotective effects in a subject in need thereof,comprising administering administrating to the subject a compoundaccording to claim 1 or a pharmaceutical composition according to claim2.
 14. A method for treating a neurodegenerative disease in a subject inneed thereof, comprising administering administrating to the subject acompound according to claim
 1. 15. The method of claim 14, wherein theneurodegenerative disease is selected from the group consisting ofmultiple sclerosis (MS), Huntington's disease (HD), spinal muscularatrophy (SMA), spinal and bullar muscular atrophy (SBMA), andamyotrophic lateral sclerosis (ALS).
 16. The compound of claim 1,wherein the formula is Formula I.
 17. The compound of claim 1, whereinthe formula is Formula II.
 18. The pharmaceutical composition of claim2, wherein the formula is Formula I.
 19. The pharmaceutical compositionof claim 2, wherein the formula is Formula II.